Project Summary/Abstract: The human pathogen, Plasmodium falciparum, the causative agent of the disease malaria, has a complex life cycle which includes development in multiple tissues within the human host and mosquito vector. During human infection, the parasite predominantly replicates asexually every 48 hours, going from one to up to 24 new parasites in every cycle. However, at a low frequency (1-3%), the parasite commits to a sexual differentiation program to produce male and female gametocytes that are critical for transmission to the mosquito vector and to complete the full lifecycle of the parasite. Development of the Plasmodium parasite within various cell types involves the regulation of nascent mRNA transcription as well as post-transcriptional mechanisms that impact mRNA stability. In most eukaryotic systems post-transcriptional regulation is mediated by the interaction of nascent mRNAs with specific RNA binding proteins (RNABPs). In this proposal we focus on the regulation of mRNA dynamics during the sexual stage of parasite development, a stage which has been relatively inaccessible to such studies until recently. By genetically engineering P. falciparum parasites to enable them to scavenge pyrimidine precursors, we can now feed them modified 4-thiouracil (4-TU), which they readily incorporate into newly transcribed RNA. These thiolated RNAs allow us to specifically address questions regarding RNA metabolism by capturing and identifying these nascent RNAs as well as their RNABPs. Our central hypothesis is that post-transcriptional mechanisms mediated by parasite specific RNA-binding proteins play a prominent role in gametocyte- specific gene regulation and are essential determinants of the parasite-transmissible stages of development. This study will directly profile real-time transcription and RNA stabilization, and will identify the RNABP involved in post-transcriptional control during the sexual stages of Plasmodium falciparum development. Aim 1 will use our 4-TU labeling method to capture sexual-stage specific mRNA dynamics throughout gametocytogenesis by measuring nascent transcription and mRNA stability beginning with commitment to gametocytogenesis. In Aim 2 4-TU labeled RNAs will be crosslinked to their cognate RNABPs and assayed by proteomics to determine an atlas of all sexual- stage RNA-binding proteins and define the RNA recognition motifs that promote this interaction. Additionally, we will use a targeted immunoprecipitation-based approach to define the specific RNA- protein interactions of several sexual-stage RNABPs including PfPuf2. This study will pave the way for future studies of RNA regulation using these tools and approaches and will enable the design of new antimalarial strategies to combat malaria transmission. !